Fig 1: δ- and ε-tubulin are enriched in the adult testis.qPCR analysis of Tubd1 (a) and Tube1 (b) mRNA transcript levels in whole testis homogenates from Katnal2WT/WT mice of various ages. Data are normalised to Hprt and presented relative to Katnal2WT/WT day 70. δ-tubulin (TUBD1) (c) and ε-tubulin (TUBE1) (e) immunolabelling (green) in Katnal2WT/WT testis sections. Examples of key cell types are indicated: pachytene spermatocytes (white arrowheads), round spermatids (magenta arrowhead), spermatid flagella (yellow arrowhead) and elongating spermatids (red arrowhead). Blue represents DNA as labelled by TOPRO. Scale bars in c and e = 10 μm. TUBD1 (d) and TUBE1 (f) localisation in Katnal2WT/WT and Katnal2Y86C/Y86C isolated elongating spermatids. Cells were counterstained with DAPI to visualise DNA, and either α-tubulin or γ-tubulin immunolabelling as markers for microtubules and centrioles, respectively. Scale bars in d and f = 2 μm. Primary antibody negative controls for (c–f) are included in S7 Fig.
Fig 2: KATNAL2 interacts with δ- and ε-tubulin.KATNAL2 interaction with δ-tubulin (TUBD1) (a) and ε-tubulin (TUBE1) (b) was indicated by co-immunoprecipitation of pEGFP-KATNAL2-pmCherry-TUBD1 and pEGFP-KATNAL2-pmCherry-TUBE1 complexes respectively, using anti-GFP beads. (c) Interactions were confirmed by co-immunoprecipitation assays from mouse testis lysate and (d) in situ proximity ligation assays. (a-b) Input: whole cell lysate from transfected cells; GFP IP: immunoprecipitation with GFP conjugated beads. (a) The left upper panel shows mCherry-TUBD1 was successfully transfected into both cell populations and the left lower panel shows EGFP and EGFP-KATNAL2 were successfully transfected into the desired cell population. The right upper panel confirmed mCherry-TUBD1 can bind to EGFP-KATNAL2, but not to EGFP. Right lower panel confirmed EGFP and EGFP-KATNAL2 proteins were successfully precipitated with GFP beads. (b) The left upper panel shows mCherry-TUBE1 was successfully transfected into both cell populations and the left lower panel shows EGFP and EGFP-KATNAL2 were successfully transfected into the desired cell population. The right upper panel confirmed mCherry-TUBE1 can bind to EGFP-KATNAL2, but not to EGFP. Right lower panel confirmed EGFP and EGFP-KATNAL2 proteins were successfully precipitated with GFP beads. (c) Input: whole testis lysate from adult mice; KATNAL2 IP: immunoprecipitation (+) and negative control immunoprecipitation (-) using an antibody directed against KATNAL2. Blots were probed with antibodies directed against KATNAL2, TUBD1 and TUBE1. (d) Representative images of in situ proximity ligation assays using antibodies directed against KATNAL2 and TUBD1 and against KATNAL2 and TUBE1 in Katnal2WT/WT isolated elongating spermatids. The specificity of labelling was assessed via the parallel labelling of Katnal2KO/KO cells. Green represents the acrosome as labelled by PNA and blue represents DNA as labelled by DAPI. Manufacturer recommended negative controls are included in S8 Fig. Scale bars in d = 2 μm.
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